Precise temporal control of mesenchymal differentiation into bone and cartilage is essential for proper development of the craniofacial skeleton. Premature differentiation within cranial sutures produces craniosynostoses whereas delayed differentiation leads to fontanel defects associated with cleidocranial and campomelic dysplasias. Thus, identifying cellular and molecular mechanisms that control the timing of skeletal differentiation is a prerequisite for preventing birth defects. Two molecules that play critical roles during skeletal differentiation are runx2 and sox9, which are required for bone and cartilage respectively. What remain unclear are mechanisms that define the temporal expression of runx2 and sox9, and establish exactly when bone and cartilage differentiate. The proposed research addresses this issue by manipulating the relative age of mesenchyme and by altering the regulation of runx2 and sox9. Quail and duck embryos have divergent growth rates and orthotopic transplants of neural crest cells destined to form the craniofacial skeleton reveal that quail donor cells differentiate into bone and cartilage earlier than duck host mesenchyme as evidenced by expression of runx2 and sox9. Three approaches are taken to test the hypothesis that neural crest mesenchyme establishes the timing of skeletal differentiation by regulating the expression of, and governing its own response to, and signals that control runx2 and sox9. Each approach involves generating chimeric embryos with either older donor mesenchyme within a relatively younger host, or younger donor mesenchyme within a relatively older host. Specific Aim 1 involves in vitro experiments to determine when tissue interactions are required for bone and cartilage formation, and to assess the extent to which neural crest cells govern these interactions. Specific Aim 2 identifies neural crest-dependent signaling events involving FGF and TGFbeta family members and their targets, which regulate runx2 and sox9 expression, and govern the timing of skeletal differentiation. Specific Aim 3 ascertains the potential of FGF and TGFbeta family members to control the timing of skeletal differentiation by employing gain- and loss-of-function approaches to regulate bone and cartilage formation. One important goal is to "rescue" the premature or delayed skeletal differentiation in chimeras, which has clear clinical implications for devising molecular-based therapies to treat disorders that affect the timing of skeletal differentiation.